Effects of carrier solutions on the viability and efficacy of canine adipose-derived mesenchymal stem cells

Background Mesenchymal stem cells (MSCs) have favorable characteristics that render them a potent therapeutic tool. We tested the characteristics of MSCs after temporal storage in various carrier solutions, such as 0.9% saline (saline), 5% dextrose solution (DS), heparin in saline, and Hartmann’s solution, all of which are approved by the U.S. Food and Drug Administration (FDA). Phosphate-buffered saline, which does not have FDA approval, was also used as a carrier solution. We aimed to examine the effects of these solutions on the viability and characteristics of MSCs to evaluate their suitability and efficacy for the storage of canine adipose-derived MSCs (cADMSCs). Results We stored the cADMSCs in the test carrier solutions in a time-dependent manner (1, 6, and 12 h) at 4 °C, and analyzed cell confluency, viability, proliferation, self-renewability, and chondrogenic differentiation. Cell confluency was significantly higher in 5% DS and lower in phosphate-buffered saline at 12 h compared to other solutions. cADMSCs stored in saline for 12 h showed the highest viability rate. However, at 12 h, the proliferation rate of cADMSCs was significantly higher after storage in 5% DS and significantly lower after storage in saline, compared to the other solutions. cADMSCs stored in heparin in saline showed superior chondrogenic capacities at 12 h compared to other carrier solutions. The expression levels of the stemness markers, Nanog and Sox2, as well as those of the MSC surface markers, CD90 and CD105, were also affected over time. Conclusion Our results suggest that MSCs should be stored in saline, 5% DS, heparin in saline, or Hartmann’s solution at 4 °C, all of which have FDA approval (preferable storage conditions: less than 6 h and no longer than 12 h), rather than storing them in phosphate-buffered saline to ensure high viability and efficacy.

Bionic Organs: Shear Forces Reduce Pancreatic Islet and Mammalian Cell Viability during the Process of 3D Bioprinting

Background: 3D bioprinting is the future of constructing functional organs. Creating a bioactive scaffold with pancreatic islets presents many challenges. The aim of this paper is to assess how the 3D bioprinting process affects islet viability. Methods: The BioX 3D printer (Cellink), 600 μm inner diameter nozzles, and 3% (w/v) alginate cell carrier solution were used with rat, porcine, and human pancreatic islets. Islets were divided into a control group (culture medium) and 6 experimental groups (each subjected to specific pressure between 15 and 100 kPa). FDA/PI staining was performed to assess the viability of islets. Analogous studies were carried out on α-cells, β-cells, fibroblasts, and endothelial cells. Results: Viability of human pancreatic islets was as follows: 92% for alginate-based control and 94%, 90%, 74%, 48%, 61%, and 59% for 15, 25, 30, 50, 75, and 100 kPa, respectively. Statistically significant differences were observed between control and 50, 75, and 100 kPa, respectively. Similar observations were made for porcine and rat islets. Conclusions: Optimal pressure during 3D bioprinting with pancreatic islets by the extrusion method should be lower than 30 kPa while using 3% (w/v) alginate as a carrier.

Filling Gaps on Stability Data: Development, Validation and Application of a Multianalyte UHPLC-DAD Method to Determine the Stability of Commonly Administered Drugs in Different Carrier Solutions Used in Palliative Care

In palliative care, continuous subcutaneous infusion (CSCI) is common practice for drug administration when oral application of drugs is not feasible or not reliable anymore. However, use of CSCI is limited to chemical stability of drugs and their combination in carrier solution. To determine the stability of different mixtures of commonly used drugs in palliative care, a multi-analyte UHPLC-DAD method controlled by an internal standard was successfully developed. The method was validated in terms of specificity, accuracy, precision, and linearity across the calibration range. Seven analytes could be separated within 10 min by C18-reversed phase chromatography. The method was successfully applied to close gaps in stability data and complete missing data for decision makers in health care units. Our results indicated the stability of binary mixtures and one ternary mixture in 0.9% saline and 5% glucose as carrier solutions. The obtained data will support pharmacists in palliative care for the preparation of parenteral drug solutions in the future.

712. Activity of Exebacase (CF-301) Against Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms on Orthopedic Kirschner Wires

Background Orthopedic foreign body-associated infection can be difficult to treat due to the formation of biofilms protecting microorganisms from both antimicrobials and the immune system. Exebacase (EXE) is a phage-derived lysin which acts as a direct lytic agent by hydrolyzing the peptidoglycan cell wall of Staphylococcus aureus. In this study, the activity of EXE was evaluated in comparison to daptomycin against MRSA biofilms on orthopedic Kirschner wires (K-wires). Methods MRSA strain IDRL-6169 was studied; it has a MIC of 0.5 µg/mL for both daptomycin (DAP) and EXE. Biofilms were formed in 1 mL of 106 cfu/mL tryptic soy broth on 0.5x0.1 mm threaded stainless steel K-wires for 10 hours, after which the wires were removed from the media and placed into 0.04 mL of either DAP or EXE at 0 (vehicle only), 0.098, 0.98, or 9.8 mg/mL. DAP+EXE was also tested, each at 0.098 mg/mL. Bacteria were quantified after 0, 2, 4, 8, and 12 hours of incubation at 37ºC. Testing was performed in triplicate. Results were reported as log10 cfu/K-wire reduction relative to vehicle alone. A 3-log10 cfu/K-wire reduction was considered bactericidal. P-values were calculated using Kruskal–Wallis. Results The bacterial burden of vehicle alone ranged from 5.49- to 6.33-log10 cfu/K-wire at all time points. Bacterial reductions for each treatment compared with carrier solution are shown in the table. DAP showed no bactericidal activity. EXE showed bactericidal activity at all concentrations at all time points studied except 0.098 mg/mL at 8 hours. There was no significant difference between EXE at 0.098 and 0.98 mg/mL at any time point but EXE at 9.8 mg/mL did show superiority over the lower concentrations. DAP+EXE 0.098 mg/mL was bactericidal at all time points. Conclusion EXE showed a rapid effect against MRSA biofilms on orthopedic K-wires apparent within the first 2 hours of exposure and was more active than daptomycin alone at the same concentrations. Disclosures All authors: No reported disclosures.

Investigation of the immobilization of probiotics as a method for their protection and delivery to the human gastrointestinal tract

The relevance of research is the experimental and analytical justification of the effectiveness of the joint use of biopolymers of animal and plant origin as a substrate in the process of immobilization of the association of probiotic cultures. Researches are executed in specialized laboratories of universities: Omsk GAU, Saratov GAU, SKFU. In the form of a substrate were used: gelatin, χ-carrageenan, low-esterified pectin, modified starch; as bioobjects are selected: L. acidophilus, B. Lactis, S. thermophilus. To obtain reliable and complete characteristics, a set of research methods was used in the work: physicochemical, sensory, and microbiological. Investigation of immobilization allowed to determine the optimal ratio of biopolymers as a carrier (substrate): pectin and gelatin, as 2:1; the total concentration of solids of the carrier solution (20.0 ± 0.5)% by weight. The total number of viable cells of probiotic microorganisms in membranes (plates) is an average of lg (11.0 ± 0.55). In order to extend the shelf life, the membranes were dried in a freeze dryer, with parameters: the temperature of the frozen product (–25 °C) and the residual pressure in the sublimate 0.013–0.133 kPa. Immobilization by microencapsulation of the association of probiotic cultures of L. acidophilus, B. Lactis and S. thermophilus into a gel of biopolymers: gelatin food, pectin gene LM 106 AS-YA, starch in a ratio of 5:1:1 was studied by microencapsulation. The obtained microcapsules were studied in imitated gastric and intestinal conditions, while the number of viable probiotic cells was determined at different times of their degradation. It was established that 20–25% of viable cells of probiotics were released from capsules in the "artificial stomach" phase, 75–80% in the "artificial bowel" phase. Innovative biotechnologies of milk based products for specialized nutrition are presented.